The invention relates to novel granulopoietic activity (GPA) proteins and nucleic acids. The invention further relates to the use of the GPA proteins in the treatment of G-CSF related disorders.
The colony stimulating factors are a class of protein hormones that stimulate the proliferation and the function of specific blood cell types such as granulocytes. Granulocytes engulf and devour microbial invaders and cell debris and thus are crucial to infection response. Granulocytes have only a 6-12 hour life span in the bloodstream and are destroyed as they function. Accordingly, it necessary for the blood marrow stem cells to rapidly and constantly generate granulocytes. Granulocyte colony stimulating factor (G-CSF) is a protein that is essential for the proliferation and differentiation of granulocytes, particularly neutrophils.
However, as a result of their fast turnover, the granulocyte count falls rapidly and markedly upon bone marrow damage, for example from treatment with traditional cancer treatments, including chemotherapeutic agents and radiation, or immunologic disorders including AIDS. Accordingly, treatment with hG-CSF has been shown to be efficacious in minimizing some of the side effects of cancer therapies, as well as in treatment of suppressed immune systems.
However, wild-type hG-CSF has several disadvantages, including storage stability problems as well as a short half-life in the blood stream.
To this end, variants of G-CSF are known; see for example U.S. Pat. Nos. 5,214,132; 5,399,345; 5,790,421; 5,581,476; 4,999,291; 4,810,643; 4,833,127; 5,218,092; 5,362,853; 5,830,705; 5,580,755; 5,399,345 and 5,416,195 and references cited therein.
However, a need still exists for proteins exhibiting both significant stability and granulopoietic activity. Accordingly, it is an object of the invention to provide granulopoietic activity (GPA) proteins, nucleic acids and antibodies for the treatment of neutrophil disorders.
In accordance with the objects outlined above, the present invention provides non-naturally occurring GPA proteins (e.g. the proteins are not found in nature) comprising amino acid sequences that are less than about 95-97% identical to hG-CSF. The GPA proteins have at least one biological property of a G-CSF protein; for example, the GPA proteins will stimulate cells with a G-CSF receptor to proliferate. Thus the invention provides GPA proteins with amino acid sequences that have at least about 5 amino acid substitutions as compared to the hG-CSF sequence shown in FIG. 1.
In a further aspect, the present invention provides non-naturally occurring GPA conformers that have three dimensional backbone structures that substantially correspond to the three dimensional backbone structure of hG-CSF. The amino acid sequence of the conformer and the amino acid sequence of the hG-CSF are less than about 95% identical. In one aspect, at least about 90% of the non-identical amino acids are in a core region of the conformer. In other aspects, the conformer have at least about 100% of the non-identical amino acids are in a core region of the conformer.
In an additional aspect, the changes are selected from the amino acid residues at positions selected from 14, 17, 20, 21, 24, 27, 28, 31, 32, 34, 38, 78, 79, 85, 89, 91, 99, 102, 103, 107, 109, 110, 113, 116, 120, 145, 146, 147, 148, 151, 153, 155, 156, 157, 160, 161, 164, 168 and 170. Preferred embodiments include at least about 5 or 10 variations.
In a further aspect, the invention provides recombinant nucleic acids encoding the non-naturally occurring GPA proteins, expression vectors comprising the recombinant nucleic acids, and host cells comprising the recombinant nucleic acids and expression vectors.
In an additional aspect, the invention provides methods of producing the GPA proteins of the invention comprising culturing host cells comprising the recombinant nucleic acids under conditions suitable for expression of the nucleic acids. The proteins may optionally be recovered.
In a further aspect, the invention provides pharmaceutical compositions comprising a GPA protein of the invention and a pharmaceutical carrier.
In an additional aspect, the invention provides methods for treating a G-CSF responsive condition comprising administering a GPA protein of the invention to a patient. The C-CSF condition may be myelo-suppresive therapy, chronic neutropenia, or peripheral blood progenitor cell collection.